Particulate Shiga Toxin 2 in Blood is Associated to the Development of Hemolytic Uremic Syndrome in Children.

Hemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children (< 3 years), is mainly related to Shiga toxins (Stx)-producing Escherichia coli (STEC) infections. STEC are confined to the gut resulting in hemorrhagic colitis, whereas Stx are delivered in blood to target kidney and brain, with unclear mechanisms, triggering HUS in 5 to 15% of infected children. Stx were found on circulating cells, free in sera (soluble Stx) or in blood cell-derived microvesicles (particulate Stx), whereby the relationship between these forms of circulating toxins is unclear. Here, we have examined 2,846 children with bloody diarrhea and found evidence of STEC infection in 5%. Twenty patients were enrolled to study the natural course of STEC infections before the onset of HUS. In patients, Stx were found to be associated to circulating cells and/or free and functionally active in sera. In most children, Stx were bound to neutrophils when high amounts of toxins were found in feces. Time-course analysis showed that Stx increased transiently in patients' sera while the decrease of toxin amount on leukocytes was observed. Notably, patients who recovered (85%) displayed different settings than those who developed HUS (15%). The distinctive feature of the latter group was the presence in blood of particulate Stx2 (Stx2 sedimented at g-forces corresponding to 1 µm microvesicles) the day before diagnosis of HUS, during the release phase of toxins from circulating cells. This observation strongly suggests the involvement of blood cell-derived particulate Stx2 in the transition from hemorrhagic colitis to HUS.

Validation on milk and sprouts of EN ISO 16654:2001 - Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Escherichia coli O157.

In 2006, the European Committee for standardisation (CEN)/Technical Committee 275 - Food analysis - Horizontal methods/Working Group 6 - Microbiology of the food chain (TC275/WG6), launched the project of validating the method ISO 16654:2001 for the detection of Escherichia coli O157 in foodstuff by the evaluation of its performance, in terms of sensitivity and specificity, through collaborative studies. Previously, a validation study had been conducted to assess the performance of the Method No 164 developed by the Nordic Committee for Food Analysis (NMKL), which aims at detecting E. coli O157 in food as well, and is based on a procedure equivalent to that of the ISO 16654:2001 standard. Therefore, CEN established that the validation data obtained for the NMKL Method 164 could be exploited for the ISO 16654:2001 validation project, integrated with new data obtained through two additional interlaboratory studies on milk and sprouts, run in the framework of the CEN mandate No. M381. The ISO 16654:2001 validation project was led by the European Union Reference Laboratory for Escherichia coli including VTEC (EURL-VTEC), which organized the collaborative validation study on milk in 2012 with 15 participating laboratories and that on sprouts in 2014, with 14 participating laboratories. In both studies, a total of 24 samples were tested by each laboratory. Test materials were spiked with different concentration of E. coli O157 and the 24 samples corresponded to eight replicates of three levels of contamination: zero, low and high spiking level. The results submitted by the participating laboratories were analyzed to evaluate the sensitivity and specificity of the ISO 16654:2001 method when applied to milk and sprouts. The performance characteristics calculated on the data of the collaborative validation studies run under the CEN mandate No. M381 returned sensitivity and specificity of 100% and 94.4%, respectively for the milk study. As for sprouts matrix, the sensitivity resulted in 75.9% in the low level of contamination samples and 96.4% in samples spiked with high level of E. coli O157 and specificity was calculated as 99.1%.

A case of haemolytic uraemic syndrome (HUS) revealed an outbreak of Shiga toxin-2-producing Escherichia coli O26:H11 infection in a nursery, with long-lasting shedders and person-to-person transmission, Italy 2015.

Purpose. Shiga toxin-producing Escherichia coli (STEC) represents a major issue for public health because of the severity of the associated illnesses, including haemolytic uraemic syndrome (HUS). In 2015, investigation of a case of HUS revealed an outbreak of Shiga toxin-2-producing E. coli O26 : H11 infection in a nursery in Italy. The investigation showed that the infection was transmitted to cases' contacts via person to person.Methods. The case finding was performed by testing for STEC stool samples of the HUS case's contacts within the family and the nursery. STEC O26 isolates were characterized by whole genome sequencing. Confirmed cases were repeatedly tested to monitor the duration of STEC shedding.Results. Eleven STEC O26 cases were identified, including adults and asymptomatic patients. Clinical illness was only observed in children. Strain characterization revealed that a single clone harbouring the stx2a and eae genes and the complete array of STEC-associated virulence genes, belonging to ST(21), was implicated in the outbreak. To reduce bacterial shedding, patients were treated with cefixime following clinical recovery. This antibiotic was well tolerated and did not induce any apparent consequences on patients' health.Conclusions. This study confirms that Stx2-producing E. coli O26 represents an emerging public health problem. The occurrence of outbreaks of infection by Stx2-producing E. coli O26 in nurseries is of particular concern, given the high probability of infection progressing in severity and resulting in secondary cases.

The Gene tia, Harbored by the Subtilase-Encoding Pathogenicity Island, Is Involved in the Ability of Locus of Enterocyte Effacement-Negative Shiga Toxin-Producing Escherichia coli Strains To Invade Monolayers of Epithelial Cells.

Locus of enterocyte effacement (LEE)-negative Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are human pathogens that lack the LEE locus, a pathogenicity island (PAI) involved in the intimate adhesion of LEE-positive strains to the host gut epithelium. The mechanism used by LEE-negative STEC strains to colonize the host intestinal mucosa is still not clear. The cell invasion determinant tia, previously described in enterotoxigenic E. coli strains, has been identified in LEE-negative STEC strains that possess the subtilase-encoding pathogenicity island (SE-PAI). We evaluated the role of the gene tia, present in these LEE-negative STEC strains, in the invasion of monolayers of cultured cells. We observed that these strains were able to invade Caco-2 and HEp-2 cell monolayers and compared their invasion ability with that of a mutant strain in which the gene tia had been inactivated. Mutation of the gene tia resulted in a strong reduction of the invasive phenotype, and complementation of the tia mutation with a functional copy of the gene restored the invasion activity. Moreover, we show that the gene tia is overexpressed in bacteria actively invading cell monolayers, demonstrating that tia is involved in the ability to invade cultured monolayers of epithelial cells shown by SE-PAI-positive E. coli, including STEC, strains. However, the expression of the tia gene in the E. coli K-12 strain JM109 was not sufficient, in its own right, to confer to this strain the ability to invade cell monolayers, suggesting that at least another factor must be involved in the invasion ability displayed by the SE-PAI-positive strains.

Molecular and phenotypic typing of enteropathogenic Escherichia coli isolated in childhood acute diarrhea in Abuja, Nigeria.

INTRODUCTION: Enteropathogenic Escherichia coli (EPEC) causes infectious diarrhea among children in developing countries. However, in Nigeria, due to limited laboratory resources, the genetic diversity of its virulence factors, which includes its intimin subtypes remain undefined. This study aims to evaluate the serotypes, antibiotic resistance and the intimin subtypes genes associated with EPEC strains isolated from children with acute diarrhea in Abuja, Nigeria. METHODOLOGY: EPEC isolates from diarrheic children aged 0-60 months in Abuja, Nigeria were analyzed. PCR for EPEC virulence gene, Hep-2 cell Adherence and serotyping were performed. EPEC strains were further subtyped by PCR reactions for the identification of intimin subtype genes: α (alpha), ß (beta), γ1 (gamma-1), and έ (epsilon). Antibiotic resistance and ESBL production was determined by CLSI guidelines. RESULTS: Overall, 18 (4.5%) out of 400 children with acute diarrhea had EPEC infection. Typical EPEC strains were detected in 14 (3.5%) whereas 4 (1.1%) were atypical EPEC. Fifteen (83.3%) of the EPEC isolated belonged to ß intimin subtype gene, while the remaining 3 EPEC isolates possessed the intimin έ subtype. No α and γ intimin subtypes were detected. Traditional EPEC serotypes O114:H14 were detected only in tEPEC strains. Marked resistant to ß-lactam agents were observed but no ESBL-producing tEPEC or aEPEC was detected. CONCLUSIONS: This is the first report of intimin subtype genes in Abuja, Nigeria. These data will be useful in facilitating the characterization of intimin variants of EPEC and some Shiga toxin-producing E. coli (STEC) in humans and other animal species.

Whole-Genome Characterization and Strain Comparison of VT2f-Producing Escherichia coli Causing Hemolytic Uremic Syndrome.

Verotoxigenic Escherichia coli infections in humans cause disease ranging from uncomplicated intestinal illnesses to bloody diarrhea and systemic sequelae, such as hemolytic uremic syndrome (HUS). Previous research indicated that pigeons may be a reservoir for a population of verotoxigenic E. coli producing the VT2f variant. We used whole-genome sequencing to characterize a set of VT2f-producing E. coli strains from human patients with diarrhea or HUS and from healthy pigeons. We describe a phage conveying the vtx2f genes and provide evidence that the strains causing milder diarrheal disease may be transmitted to humans from pigeons. The strains causing HUS could derive from VT2f phage acquisition by E. coli strains with a virulence genes asset resembling that of typical HUS-associated verotoxigenic E. coli.

Community-wide outbreak of haemolytic uraemic syndrome associated with Shiga toxin 2-producing Escherichia coli O26:H11 in southern Italy, summer 2013.

In summer 2013, an excess of paediatric cases of haemolytic uraemic syndrome (HUS) in a southern region of Italy prompted the investigation of a community-wide outbreak of Shiga toxin 2-producing Escherichia coli (STEC) O26:H11 infections. Case finding was based on testing patients with HUS or bloody diarrhoea for STEC infection by microbiological and serological methods. A case-control study was conducted to identify the source of the outbreak. STEC O26 infection was identified in 20 children (median age 17 months) with HUS, two of whom reported severe neurological sequelae. No cases in adults were detected. Molecular typing showed that two distinct STEC O26:H11 strains were involved. The case-control study showed an association between STEC O26 infection and consumption of dairy products from two local plants, but not with specific ready-to-eat products. E.coli O26:H11 strains lacking the stx genes were isolated from bulk milk and curd samples, but their PFGE profiles did not match those of the outbreak isolates. This outbreak supports the view that infections with Stx2-producing E. coli O26 in children have a high probability of progressing to HUS and represent an emerging public health problem in Europe.

Rapid Detection and Isolation of Escherichia coli O104:H4 from Milk Using Monoclonal Antibody-coated Magnetic Beads.

Monoclonal antibodies (MAbs) specific for the lipopolysaccharide (LPS) of Escherichia coli O104:H4 were produced by fusion of Sp2/O-Ag-14 mouse myeloma cells with spleen cells of Balb/c mice, immunized with heat-inactivated and sonicated E. coli O104:H4 bacterial cells. Four MAbs specific for the E. coli O104:H4 LPS (1E6G6, 1F4C9, 3G6G7, and 4G10D2) were characterized and evaluated for the use in a method for the detection of E. coli O104:H4 in milk samples that involves antibody conjugation to magnetic microbeads to reduce time and increase the efficiency of isolation. MAb 1E6G6 was selected and coupled to microbeads, then used for immuno-magnetic separation (IMS); the efficiency of the IMS method for E. coli O104:H4 isolation from milk was evaluated and compared to that of the EU RL VTEC conventional culture-based isolation procedure. Milk suspensions also containing other pathogenic bacteria that could potentially be found in milk (Campylobacter jejuni, Listeria monocytogenes, and Staphylococcus aureus) were also tested to evaluate the specificity of MAb-coated beads. Beads coated with MAb 1E6G6 showed a good ability to capture the E. coli O104:H4, even in milk samples contaminated with other bacteria, with a higher number of E. coli O104:H4 CFU reisolated in comparison with the official method (121 and 41 CFU, respectively, at 10(3) E. coli O104:H4 initial load; 19 and 6 CFU, respectively, at 10(2) E. coli O104:H4 initial load; 1 and 0 CFU, respectively, at 10(1) E. coli O104:H4 initial load). The specificity was 100%.

Development of a High Resolution Virulence Allelic Profiling (HReVAP) Approach Based on the Accessory Genome of Escherichia coli to Characterize Shiga-Toxin Producing E. coli (STEC).

Shiga-toxin producing Escherichia coli (STEC) strains possess a large accessory genome composed of virulence genes existing in multiple allelic variants, which sometimes segregate with specific STEC subpopulations. We analyzed the allelic variability of 91 virulence genes of STEC by Real Time PCR followed by melting curves analysis in 713 E. coli strains including 358 STEC. The 91 genes investigated were located on the locus of enterocyte effacement (LEE), OI-57, and OI-122 pathogenicity islands and displayed a total of 476 alleles in the study population. The combinations of the 91 alleles of each strain were termed allelic signatures and used to perform cluster analyses. We termed such an approach High Resolution Virulence Allelic Profiling (HReVAP) and used it to investigate the phylogeny of STEC of multiple serogroups. The dendrograms obtained identified groups of STEC segregating approximately with the serogroups and allowed the identification of subpopulations within the single groups. The study of the allelic signatures provided further evidence of the coevolution of the LEE and OI-122, reflecting the occurrence of their acquisition through a single event. The HReVAP analysis represents a sensitive tool for studying the evolution of LEE-positive STEC.

Whole genome sequence comparison of vtx2-converting phages from Enteroaggregative Haemorrhagic Escherichia coli strains.

BACKGROUND: Enteroaggregative Haemorrhagic E. coli (EAHEC) is a new pathogenic group of E. coli characterized by the presence of a vtx2-phage integrated in the genomic backbone of Enteroaggregative E. coli (EAggEC). So far, four distinct EAHEC serotypes have been described that caused, beside the large outbreak of infection occurred in Germany in 2011, a small outbreak and six sporadic cases of HUS in the time span 1992-2012. In the present work we determined the whole genome sequence of the vtx2-phage, termed Phi-191, present in the first described EAHEC O111:H2 isolated in France in 1992 and compared it with those of the vtx-phages whose sequences were available. RESULTS: The whole genome sequence of the Phi-191 phage was identical to that of the vtx2-phage P13374 present in the EAHEC O104:H4 strain isolated during the German outbreak 20 years later. Moreover, it was also almost identical to those of the other vtx2-phages of EAHEC O104:H4 strains described so far. Conversely, the Phi-191 phage appeared to be different from the vtx2-phage carried by the EAHEC O111:H21 isolated in the Northern Ireland in 2012.The comparison of the vtx2-phages sequences from EAHEC strains with those from the vtx-phages of typical Verocytotoxin-producing E. coli strains showed the presence of a 900 bp sequence uniquely associated with EAHEC phages and encoding a tail fiber. CONCLUSIONS: At least two different vtx2-phages, both characterized by the presence of a peculiar tail fiber-coding gene, intervened in the emergence of EAHEC. The finding of an identical vtx2-phage in two EAggEC strains isolated after 20 years in spite of the high variability described for vtx-phages is unexpected and suggests that such vtx2-phages are kept under a strong selective pressure.The observation that different EAHEC infections have been traced back to countries where EAggEC infections are endemic and the treatment of human sewage is often ineffective suggests that such countries may represent the cradle for the emergence of the EAHEC pathotype. In these regions, EAggEC of human origin can extensively contaminate the environment where they can meet free vtx-phages likely spread by ruminants excreta.

Shiga toxin-converting phages and the emergence of new pathogenic Escherichia coli: a world in motion.

Shiga toxin (Stx)-producing Escherichia coli (STEC) are pathogenic E. coli causing diarrhea, hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). STEC are characterized by a constellation of virulence factors additional to Stx and have long been regarded as capable to cause HC and HUS when possessing the ability of inducing the attaching and effacing (A/E) lesion to the enterocyte, although strains isolated from such severe infections sometimes lack this virulence feature. Interestingly, the capability to cause the A/E lesion is shared with another E. coli pathogroup, the Enteropathogenic E. coli (EPEC). In the very recent times, a different type of STEC broke the scene causing a shift in the paradigm for HUS-associated STEC. In 2011, a STEC O104:H4 caused a large outbreak with more than 800 HUS and 50 deaths. Such a strain presented the adhesion determinants of Enteroaggregative E. coli (EAggEC). We investigated the possibility that, besides STEC and EAggEC, other pathogenic E. coli could be susceptible to infection with stx-phages. A panel of stx2-phages obtained from STEC isolated from human disease was used to infect experimentally E. coli strains representing all the known pathogenic types, including both diarrheagenic E. coli (DEC) and extra-intestinal pathogenic E. coli (ExPEC). We observed that all the E. coli pathogroups used in the infection experiments were susceptible to the infection. Our results suggest that the stx2-phages used may not have specificity for E. coli adapted to the intestinal environment, at least in the conditions used. Additionally, we could only observe transient lysogens suggesting that the event of stable stx2-phage acquisition occurs rarely.

Identification of two allelic variants of toxB gene and investigation of their distribution among Verocytotoxin-producing Escherichia coli.

Verocytotoxin-producing Escherichia coli (VTEC) are food borne pathogens causing severe human infections. The virulence genes asset of VTEC is complex and has not been completely defined yet. Nonetheless, all the virulence genes described so far have been described as conveyed by mobile genetic elements. A gene, termed toxB, has been identified in a large virulence plasmid of VTEC O157, later described in similar plasmids carried by VTEC O26 and O145. In this study we identified for the first time an intact copy of toxB gene in a plasmid present in a VTEC O111 strain and observed the existence of two allelic variants of the gene, that we termed toxB1 and toxB2. We investigated the distribution of the two alleles in a panel of VTEC strains belonging to different serogroups and demonstrated that this gene is present only in VTEC serogroups associated with the most severe forms of the infections such as those belonging to the five serogroups O157, O26, O111, O103 and O145 and that the two alleles segregate with the serogroup of the hosting strains. In particular the toxB1 variant was only present in VTEC O157 while the toxB2 allele was present in the remaining four VTEC serogroups.

Identification and characterization of a peculiar vtx2-converting phage frequently present in verocytotoxin-producing Escherichia coli O157 isolated from human infections.

Certain verocytotoxin-producing Escherichia coli (VTEC) O157 phage types (PTs), such as PT8 and PT2, are associated with severe human infections, while others, such as PT21, seem to be restricted to cattle. In an attempt to delve into the mechanisms underlying such a differential distribution of PTs, we performed microarray comparison of human PT8 and animal PT21 VTEC O157 isolates. The main differences observed were in the vtx2-converting phages, with the PT21 strains bearing a phage identical to that present in the reference strain EDL933, BP933W, and all the PT8 isolates displaying lack of hybridization in some regions of the phage genome. We focused on the region spanning the gam and cII genes and developed a PCR tool to investigate the presence of PT8-like phages in a panel of VTEC O157 strains belonging to different PTs and determined that a vtx2 phage reacting with the primers deployed, which we named Φ8, was more frequent in VTEC O157 strains from human disease than in bovine strains. No differences were observed in the production of the VT2 mRNA when Φ8-positive strains were compared with VTEC O157 possessing BP933W. Nevertheless, we show that the gam-cII region of phage Φ8 might carry genetic determinants downregulating the transcription of the genes encoding the components of the type III secretion system borne on the locus of enterocyte effacement pathogenicity island.

Public Health Microbiology of Shiga Toxin-Producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) strains are the only pathogenic group of E. coli that has a definite zoonotic origin, with ruminants and, in particular, cattle being recognized as the major reservoir. Most human STEC infections are food borne, but the routes of transmission include direct contact with animals and a variety of environment-related exposures. Therefore, STEC public health microbiology spans the fields of medical, veterinary, food, water, and environmental microbiology, requiring a "One Health" perspective and laboratory scientists with the ability to work effectively across disciplines. Public health microbiology laboratories play a central role in the surveillance of STEC infections, as well as in the preparedness for responding to outbreaks and in providing scientific evidence for the implementation of prevention and control measures. This article reviews (i) how the integration of surveillance of STEC infections and monitoring of these pathogens in animal reservoirs and potential food vehicles may contribute to their control; (ii) the role of reference laboratories, in both the public health and veterinary and food sectors; and (iii) the public health perspectives, including those related to regulatory issues in both the European Union and the United States.

Enterohemorrhagic Escherichia coli O26:H11/H-: a new virulent clone emerges in Europe.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) O26 causes diarrhea and hemolytic uremic syndrome (HUS). Strains harboring the stx1a gene prevail, but strains with stx2a as the sole Shiga toxin-encoding gene are now emerging. The traits and virulence of the latter set of strains are unknown. We correlated stx genotypes of 272 EHEC O26 strains isolated in 7 European countries between 1996 and 2012 with disease phenotypes. We determined phylogeny, clonal structure, and plasmid gene profiles of the isolates and portray geographic and temporal distribution of the different subgroups. METHODS: The stx genotypes and plasmid genes were identified using polymerase chain reaction, phylogeny was assigned using multilocus sequence typing, and clonal relatedness was established using pulsed-field gel electrophoresis. RESULTS: Of the 272 EHEC O26 isolates, 107 (39.3%), 139 (51.1%), and 26 (9.6%) possessed stx1a, stx2a, or both genes, respectively. Strains harboring stx2a only were significantly associated with HUS (odds ratio, 14.2; 95% confidence interval, 7.9-25.6; P < .001) compared to other stx genotypes. The stx2a-harboring strains consist of 2 phylogenetically distinct groups defined by sequence type (ST) 21 and ST29. The ST29 strains are highly conserved and correspond by plasmid genes to the new virulent clone of EHEC O26 that emerged in Germany in the 1990s. This new clone occurred in 6 of the 7 countries and represented approximately 50% of all stx2a-harboring EHEC O26 strains isolated between 1996 and 2012. CONCLUSIONS: A new highly virulent clone of EHEC O26 has emerged in Europe. Its reservoirs and sources warrant identification.

Multicenter evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx nomenclature.

When Shiga toxin-producing Escherichia coli (STEC) strains emerged as agents of human disease, two types of toxin were identified: Shiga toxin type 1 (Stx1) (almost identical to Shiga toxin produced by Shigella dysenteriae type 1) and the immunologically distinct type 2 (Stx2). Subsequently, numerous STEC strains have been characterized that express toxins with variations in amino acid sequence, some of which confer unique biological properties. These variants were grouped within the Stx1 or Stx2 type and often assigned names to indicate that they were not identical in sequence or phenotype to the main Stx1 or Stx2 type. A lack of specificity or consistency in toxin nomenclature has led to much confusion in the characterization of STEC strains. Because serious outcomes of infection have been attributed to certain Stx subtypes and less so with others, we sought to better define the toxin subtypes within the main Stx1 and Stx2 types. We compared the levels of relatedness of 285 valid sequence variants of Stx1 and Stx2 and identified common sequences characteristic of each of three Stx/Stx1 and seven Stx2 subtypes. A novel, simple PCR subtyping method was developed, independently tested on a battery of 48 prototypic STEC strains, and improved at six clinical and research centers to test the reproducibility, sensitivity, and specificity of the PCR. Using a consistent schema for nomenclature of the Stx toxins and stx genes by phylogenetic sequence-based relatedness of the holotoxin proteins, we developed a typing approach that should obviate the need to bioassay each newly described toxin and that predicts important biological characteristics.

Pathogenic potential to humans of bovine Escherichia coli O26, Scotland.

Escherichia coli O26 and O157 have similar overall prevalences in cattle in Scotland, but in humans, Shiga toxin-producing E. coli O26 infections are fewer and clinically less severe than E. coli O157 infections. To investigate this discrepancy, we genotyped E. coli O26 isolates from cattle and humans in Scotland and continental Europe. The genetic background of some strains from Scotland was closely related to that of strains causing severe infections in Europe. Nonmetric multidimensional scaling found an association between hemolytic uremic syndrome (HUS) and multilocus sequence type 21 strains and confirmed the role of stx(2) in severe human disease. Although the prevalences of E. coli O26 and O157 on cattle farms in Scotland are equivalent, prevalence of more virulent strains is low, reducing human infection risk. However, new data on E. coli O26-associated HUS in humans highlight the need for surveillance of non-O157 enterohemorrhagic E. coli and for understanding stx(2) phage acquisition.

Virulotyping of Salmonella enterica serovar Napoli strains isolated in Italy from human and nonhuman sources.

Salmonella enterica serovar Napoli is an emerging serovar in Italy, France, and Switzerland, but little is known about its pathogenicity to humans. A collection of 112 strains of Salmonella Napoli isolated in Italy from human cases, foods of animal origin, and the environment have been characterized by the detection of a set of virulence genes, pulsed-field gel electrophoresis (PFGE), and antibiotic susceptibility. All the strains examined were susceptible to all the antimicrobials tested. The Salmonella pathogenicity islands genes ssaQ, mgtC spi_4D, and sopB were present from 75.0% to 100% of the tested strains. Only one human and four environmental strains showed the avrA gene. The phage-related sopE1 gene was present in 93% of the strains, whereas sodC1 and gipA genes were only in four and two environmental strains, respectively. The bcfC fimbrial gene was present in all the animal/food strains, in the 71.4% of environmental strains, and in 46.8% of the human strains, respectively. Overall, we observed 10 distinct virulence profiles (VP), but VP1-2-3 included 99 out of 112 strains. PFGE showed that 103 out of 111 isolates were grouped in four major clusters and three minor clusters, whereas two strains were totally unrelated. The most represented PFGE clusters mainly correlated with the virulotypes are VP1, VP2, and VP3. Salmonella Napoli shows an array of virulence genes similar to other serovars considered of public health importance and confirming its capability to cause infection in human. Concerning possible source of infection or reservoirs, the results did not point out any, but our hypothesis is that the environment can act as the main reservoir for Salmonella Napoli, and from there it can spill over to animals and humans. Further studies are needed to increase the knowledge on the ecology of Salmonella Napoli serovar and on the main risk factors for human infection.

Use of antimicrobials in companion animal practice: a retrospective study in a veterinary teaching hospital in Italy.

OBJECTIVES: To describe the use of antimicrobials in a veterinary teaching hospital for companion animals in Italy, with particular regard to the agreement with recommendations of prudent use METHODS: The study was conducted with a retrospective, cross-sectional design. The population under investigation included 18,905 cats and dogs that were referred to the hospital between 2000 and 2007. Two different samples of the clinical paper forms were randomly selected to estimate the prevalence of animals receiving an antimicrobial prescription and to describe the pattern of antimicrobials used in relation to the condition being treated. The proportion of antimicrobials prescribed accomplishing recommendations of prudent use was also estimated, as well as the level of agreement with specific, diagnosis-based guidelines for antimicrobial use. RESULTS: Broad-spectrum antimicrobials, including penicillins with ß-lactamase inhibitors, first-generation cephalosporins and fluoroquinolones, were the most frequently prescribed compounds. Antimicrobials prescribed with the support of microbiological analyses and susceptibility testing were less than 5%. Among the recommendation of prudent use, the availability of information from laboratory testing had the poorest degree of agreement, while the other evaluated items were accomplished in most of the cases. CONCLUSIONS: Our results highlight the need to improve the procedures of antimicrobial prescription in the study setting. This can be achieved by supporting the guidance for antimicrobial use at the local level, with the adoption of specific guidelines, and at the national level with a further implementation of the policies of prudent prescriptions.